Method of genetic testing

ABSTRACT

Elevated levels of Inhibin-A in maternal serum or plasma have been shown to indicate the presence of Down&#39;s Syndrome. The assay method comprises the use of a monoclonal antibody specific for at least part of the inhibin-A β sub-unit (βA), and another monoclonal antibody specific for at least part of the Inhibin-A α sub-unit. The βA antibody is used to capture Inhibin-A from the test sample, and the α sub-unit antibody is used as the detection antibody and is linked to a detectable marker. The method is carried out in the first or second trimester as a screening test to select patients for subsequent diagnostic testing.

This invention relates to the detection of possible geneticabnormalities and more particularly to those of Down's Syndrome.

Prenatal screening for Down's syndrome has become an important andestablished part of modern antenatal care. At present, most screeningprogrammes depend upon maternal age in combination with the measurementof human chorionic gonadotrophin (hCG) and α feto-protein (AFP), with orwithout unconjugated oestriol (uE3), in maternal serum at 16 weeksgestation. Such an approach will detect approximately 65% of Down'saffected pregnancies for an amniocentesis rate of 5%. However, it haslong been hoped that improvements in screening might increase thisdetection rate, while minimising the amniocentesis rate, and also allowtesting to be performed earlier in pregnancy. To this end, it wasrecently reported that inhibin, a heterodimeric glycoprotein produced byvarious tissues, including the placenta, was elevated in the secondtrimester in maternal serum from Down's syndrome pregnancies compared tonormal pregnancies (van Lith et al. 1992. Second-trimester maternalserum immunoreactive inhibin as a marker for fetal Down's syndrome,Prenatal Diagnosis 12, 801-806; and Spencer, K. et al. 1993. Elevatedlevels of maternal serum inhibin immunoreactivity in second trimesterpregnancies affected by Down's syndrome, Annals of Clinical Biochemistry30, 219-220).

In addition, a number of fetal and placental proteins have undergoneinitial evaluation of their usefulness as first, rather than second,trimester maternal serum markers for Down's syndrome (Macintosh M. C. M.and Chard, T., 1993, Biochemical screening for Down's syndrome in thefirst trimester of pregnancy, Fetal and Maternal Medicine Reviews 5,181-190 and references therein).

Screening earlier in pregnancy, in the first trimester, would haveconsiderable advantages over the current second trimester timing.Several maternal serum markers, including α fetoprotein (AFP), b-subunitof human chorionic gonadotrophial (β-hCG). Pregnancy-associatedplacental protein A (PAPP-A) have been assessed as first trimestermarkers of which β-hCG appears the most promising of these to date. Acombination of maternal age, β-hCG and AFP has been predicted to otter adetection rate of 54% for an amniocentesis rate (or false positiverate--FPR) of 5% (Aitken et al, 1993, Biochemical screening forchromosome abnormalities and neutral tube defects in the firsttrimester, Journal of Medical Genetics 30,336). Clearly, there is stilla need for a more reliable test with improved sensitivity andspecificity which would preferably be applicable to both the first orsecond trimester, but especially the former.

It has now been established that enhanced levels of a particular speciesof the hormone Inhibin in maternal body fluids are indicative of thepossible presence of fetal abnormality of this kind.

The hormone Inhibin is known to be involved in human and animalreproduction although its precise role is not yet established. It is nowknown that there is a "family" of inhibins known as Inhibin A andInhibin B. These molecules are dimers composed of two sub-unit monomers.Inhibin-A consists of two protein sub-units, termed α and βA, which arejoined together by disulphide bonds.

European patent 185,034, dating from applications in 1984 and 1985,precedes the discovery of a family of inhibins and discloses one dimericform of inhibin, which it identifies in terms of molecular weight,sub-unit structure, and other properties. EP 185,034 sizes one sub-unitat molecular weight 14,000+/-2000 kilodaltons (12K or 14K) and thelarger sub-unit as a 44K molecule. Later research suggests that theprimary active form of dimeric inhibin-A in biological fluids is of size32K, corresponding to a 12K βA and a 20K α sub-unit. The 32K inhibin isthe mature form produced by post-translational processing of precursorforms of molecular weight 65K and 56K. Immunoreactive α monomer alsocirculates in the body, a factor which complicates the assay of inhibin.It is common to refer to "inhibin forms" as signifying the totality ofcirculating inhibin proteins.

The complex nature of Inhibin and the fact that both monomeric anddimeric forms circulate in the body presents formidable problems ofanalytical determination and biological interpretation of researchresults. For example, previously reported immunological methods ofmeasurement of Inhibin have not enabled clear conclusions to be madebecause immunoreactivity measurement does not discriminate between thevarious molecular species described above, and is therefore not areliable guide to functional bioactivity. The Van Lith et al and Spenceret al publications mentioned above are the only two publicationsdescribing the use of immunoreactive Inhibin in the detection of Down'ssyndrome in the second trimester. Both of these studies utilised acommercial enzymometric assay (Medgenix, High Wycombe) with two anti αsubunit antibodies. Both studies reported poor discrimination due to awide distribution of results in both control and Down's syndromesamples.

The present invention comprises a method of testing for indications ofDown's syndrome which comprises measuring the level of Inhibin-A inmaternal body fluid preferably by means of an assay which is specificfor Inhibin-A.

In accordance with the invention, levels of all molecular forms ofdimeric inhibin-A are preferably measured, irrespective of molecularsize. However, we do not preclude the possibility that it may besufficient for the purposes of the present invention to assayspecifically for the mature 32K form of the dimeric protein.

Any convenient and accurate method of assay of dimeric inhibin-A may beused for the diagnostic purposes of this invention. Various assays areknown but some, in their present form described in the literature, areless suitable than the preferred assay which will be described in detailhereinafter. For example Baly et al recently reported development oftwo-site assays for various inhibin forms using monoclonal andpolyclonal antibodies raised to recombinant inhibin. (Baly, D. L.,Aldisim, D. F., Krummer, A., Woodruff, T. K., Soules, M. R., Chen, S.A., Fendly, B. M., Bald, L. N., Mathet, J. P. & Lucas, C. (1993)Development of a specific and sensitive two-site enzyme-linkedimmunoadsorbent assay for measurement of inhibin-A in serum.Endocrinology, 132, 2099-2103). However, the detection limit of theirassay for dimeric inhibin-A was 1000 pg/ml of serum, and in the datapresented, inhibin could not be detected in all the samples, even fromwomen undergoing gonadotrophin therapy.

The preferred assay is one based on monoclonal antibody technology. Thusthe assay method may comprise the use of antibodies specific for atleast part of the Inhibin-A α and βA sub-units.

The βA antibody is preferably derived from a hybridoma prepared using asimmunogen a synthetic peptide corresponding to a part of the βAsub-unit. The α sub=unit antibody is preferably derived from a hybridomaprepared using as immunogen a synthetic peptide corresponding to a partof the α sub-unit. Advantageously, the βA antibody is used to captureInhibin from the test sample and the α sub-unit antibody is used as thedetection antibody and is linked to a detectable marker e.g. an enzyme.Fragments of these antibodies having the same specificity as the wholeantibodies may also be used. The term `antibody` is used herein toinclude such fragments.

The antibodies used in the assay are derived from hybridomas produced bymethods which have been described in the literature (Groome et al 1990Monoclonal and polyclonal antibodies reactive with the 1-32 aminoterminal peptide of 32K human inhibin. Hybridoma, 9, 31-41; and Groome,et al, 1991, Preparation of monoclonal antibodies reacting with thebeta-A subunit of human ovarian inhibin. Hybridoma, 10, 309-316). Theassay has also been described by Groome, et al, 1993, Immunoassays forinhibin and its subunits. Further applications of the synthetic peptideapproach, J. Immunol. Methods 165, 167). These Groome et al publicationsare incorporated herein by reference.

The preferred assay procedure, to avoid interference from heterophileantibodies, is to use the Fab fragment of the monoclonal antibody to theinhibin α sub-unit attached to an enzyme rather than the whole antibodymolecule. The inclusion of 5% (v/v) mouse serum and 5% w/v) Triton×100in the assay diluent are also important in maintaining the specificityof the assay.

The antibodies and a detailed protocol are available from Serotec Ltd.22 Hankside, Station Approach, Kidlington, Oxford, OX5 1JE.

The immobilization of the capture antibody (that to the β A sub-unit) onhydrazide plates (Avidplate-HZ from Bioprobe) rather than by passiveadsorption on ordinary microplates is important in retaining a highdensity of functional antibody essential to give good recoveries ofinhibin from serum and plasma. In an earlier version of the assay itappeared that other methods of immobilizing the anti-β A antibody gavesimilar results for standards diluted in assay buffer. When these werecompared with the hydrazide plates procedure the latter gave much betterrecoveries of recombinant inhibin spiked into human sera.

SPECIFICITY OF THE ASSAY

Until recently it was not clear whether the assay as described would beable to distinguish inhibin-A from inhibin B. The synthetic peptidesequence from the human β A sub-unit used as an immunogen to make the E4antibody has homology with the corresponding sequence from the beta Bsub-unit and the two peptides both react well with the antibody.Recently with samples of recombinant materials we have established thatinhibin-B, activin A and activin B all have very low cross-reactivity inour assay as shown below.

    ______________________________________    Percentage cross reaction    ______________________________________           Inhibin-A                  100           Inhibin-B                  0.012           Activin A                  <0.002           Activin B                  >0.001    ______________________________________

Thus this assay in practice can be regarded as measuring specificallyforms of inhibin-A in human serum and plasma samples. The assay does notdetect free forms of the α-sub-unit.

The method of this invention may be used with appropriate maternal bodyfluids e.g. serum or plasma. The method is effective when carried out inthe first or second trimester. The method may be viewed as a screeningtest to select patients for subsequent diagnostic testing. In Down'ssyndrome pregnancies, the median level of inhibin-A was noted to be morethan twice that in normal pregnancies. Importantly, the degree ofdifference was maintained across both first and second trimesters. Thisis the first report of the use of a dimeric inhibin-A assay in pregnancyand the subsequent successful application of the assay to the detectionof Down's syndrome in the first and second trimester of pregnancy. Itwill be shown that using the measurement of inhibin-A alone in the firsttrimester, detection rates that compare very favourably with currentlyavailable combinations are possible. Also, in the second trimester whenused alone inhibin-A offers high rates of detection and when combinedwith current screening will significantly improve detection rates. Priorto describing these data in detail, the methodology pertaining to theproduction of monoclonal antibodies and to the assay will be described.

As indicated previously, the production of hybridoma cell lines whichsecrete the antibodies has been described in the literature. Furtherdirections for preparing the most effective cell-lines are described inthe following section.

Factors important to success in making a high affinity antibody usefulfor immunoassay

The capture antibody is required to have a very high affinity forinhibin, as evidenced by its performance in the two-site assay fordimeric inhibin described later. The affinity of the antibody is furtherincreased when the inhibin in standards and samples is preoxidised withhydrogen peroxide. This oxidises two methionine residues in the regionof the epitope. (Knight, P. G. and Muttukrishna, S. (1993) Measurementof dimeric inhibin using a modified two-site IRMA specific for oxidised(met 0) inhibin Poster at Serono symposium on Inhibin andInhibin-related substances, Siena June 1993).

Most anti-peptide antibodies raised to internal peptide sequences reactwith the parent protein with low affinity. The important factors are:

1. The frequency of clones making such good antibodies is low.

2. It is recommended to generate as many peptide-reactive clones aspossible and to screen rigorously on the native molecule to selectclones which react with the whole molecule.

3. Four intravenous doses of tuberculin/peptide conjugate should begiven on each of the four days before spleen cell removal. We haveroutinely used up to 200 micrograms of peptide per injection and havesupplemented this with intraperitoneal injections on occasions when thesuccess of the intravenous injection was in doubt. The spleen ofimmunized mice is invariably large indicating hyperimmunization.

4. We have noted a particular value to tuberculin as a carrier forsynthetic peptide immunizations in mice primed three weeks earlier with1 dose of human BCG vaccine (Glaxo) given subcutaneously. The advantageis that BCG vaccination gives a high frequency of helper T cellsrecognizing tuberculin without generating a lot of irrelevant B cellswhich go on to give unwanted hybridomas. With conventional carriersafter an aggressive immunization scheme such as that described most ofthe clones growing after the fusion would be to the carrier making itmuch harder to identify the clones making antibody to the peptideimmunogen.

The following materials and methods have been used.

Preparation of Antibody to βA sub-unit (E4)

Coupling of Peptides to carrier protein

The location on the human inhibin β A inhibin subunit of the sevenpeptides used for immunization are shown in FIG. 1 of Groome et alHybridoma 10 309-316 (1991). The peptides were made using Fmoc chemistryand contained a single cysteine residue which was either part of thenatural sequence or added only to facilitate coupling. Coupling of eachpeptide to tuberculin via the --SH group was made usingheterobifunctional ester using protocols obtained in a kit availablefrom Cambridge Research Biochemicals, Button End Industrial Estate,Harston, cambridge, U.K. or by other well-known methods.

Immunization and Screening of mice

Male Balb/c mice were initially primed with one dose of human BCGvaccine (Glaxo). Mice then received four monthly subcutaneous boosts of50 micrograms of each peptide as a tuberculin conjugate in Freund'sincomplete adjuvant. Six mice were immunised with each peptide as atuberculin conjugate. When tested by ELISA on plates coated with bovine32 KDa, inhibin peptides 1, 3, 4 and 5 gave no responses distinguishablefrom unimmunized mice. Peptide 6 produced one medium responder (A=0.4),peptide 2 produced two medium responders (A=0.4 and A=0.8) and peptide 9produced two high responders (Absorbance greater than 2) and four mediumresponders (A=0.9, 1.0, 1.4, 0.7). The highest responding mouse wasassessed by ELISA and each of the last four days before fusion itreceived 50 micrograms of peptide as a tuberculin conjugateintravenously in saline.

ELISA screening protocols (test for antibodies)

Purified 32 KDa bovine inhibin was purchased from Peninsula laboratoriesand covalently attached to Cobind (Polyfiltronics) plates according tothe manufacturers instructions. A coating concentration of 0.1micrograms per ml was used in phosphate buffered saline (PBS) for 5 hrsat 37° C. These plates are no longer available but similar results havebeen obtained with maleic anhydride activated plates (Pierce Chemicals).Excess sites on the plastic were blocked using 1% (w/v) bovine serumalbumin in PBS for 1 hr at room temperature. Plates were then washed tentimes with 0.05% (w/v) TWEEN 80 in 0.15 mol/1 sodium chloride. TWEEN isthe trade name of a series of general-purpose emulsifiers and surfaceactive agents that are polyoxyethylene derivatives of fatty acid partialesters of sorbitol and hydrides. Similar results have been obtained withmaleic anhydride activated plates (Pierce Chemicals).

Mouse sera to be tested were diluted 1 in 1000 in diluent consisting of0.5% (w/v) TWEEN 80.0.15 mol/1 sodium chloride, 0.05 mol/1 phosphatebuffer pH 7 and 1% (w/v) bovine serum albumin diluent. Hybridomasupernatants for screening were diluted 1 in 2 in this diluent. Primaryantibody binding was allowed to take place for 18 hr at roomtemperature. Then the plate was washed and peroxidase labelledanti-mouse IgG/peroxidase conjugate (1 in 1000) was added for 30 mins.After a final wash 0.1 ml of peroxidase TMB substrate (Dynatek) wasadded. Absorbances were read on a Dynatek mini reader of 405 nm after a1 hr incubation period.

Cell Fusion

One of the two high responding mice immunized with peptide 9 was boostedintravenously and the spleen used to carry out a fusion. The mouse wasinjected IV with peptide 9/Tuberculin on each of the 4 days beforefusion. The fusion was plated out over 20 microplates (1920) wells).After about 10 days, there were 1-3 clones growing in each well. Afterscreening by ELISA on plates coated with 32 KDa inhibin only eightpositives were observed. Eventually six stable clones were obtained.Five of these were isotyped as 1 gM and the remaining clone designatedE4 made an IgG2b. Clone E4 could be grown as ascites with 1.5-2 mg/ml ofspecific antibody. The antibody could be purified on protein A withoutloss of activity provided it was promptly neutralised after elution atpH 3.5. Growth in a hollow fibre bioreator produced 2 mg/ml of antibodyand the clone was stable.

Immunoconcentrations

Inhibin forms were concentrated from 200 ml of bovine follicular fluidusing the monoclonal antibody to the α subunit (see later). The antibodywas attached to Affigel. The inhibin forms were eluted to 6 Mguinidine-HCl, dialysed against 0.063 mol/1 Tris-Cl pH 6.75, and thentreated with 1% (w/v) sodium dodecyl sulphate and 1 (w/v)mercaptoethanol before heating at 100 degrees for 5 minutes. Thefollicular fluid forms from 200 ml of follicular fluid were eventuallyin 4 ml of solution.

Electrophoresis and blotting

One microliter samples of the reduced and denatured preparation wereelectrophoresed on a pre-prepared 20% (w/v) homogenous sodium dodecylsulphate polyacrylamide gels obtained from Pharmacia. Electrophoresiswas carried out on a Pharmacia Phastsystem. In each run biotinylatedmolecular weight markers from Sigma were used alongside the inhibinpreparations. After electrophoresis Phastsystem was used to transfer ablot of the gels onto nitrocellulose by diffusion blotting at elevatedtemperature as described in the Phastsystem manual. Subsequently, theblot was developed by successive incubations either with clone E4 (madehere and reactive with the β subunit), or clone R1 (made previously andreactive with the α subunit; After this, followed biotinylatedanti-mouse IgG (Serotec) and a histochemical stain for the location ofalkaline phosphatase activity. Molecular weight calculations were madefrom enlarged photographs of the blots. The monoclonal antibody fromclone E4 gave two bands corresponding to two different molecular weightforms of the β-A subunit.

Inhibition ELISA

A concentration of clone E4 antibody sufficient to produce in the ELISAas an absorbance of 0.6 was preincubated overnight at 4° C. with variousdilutions of bovine follicular fluid prior to ELISA to determine whetherthe monoclonal antibody would react with the forms of inhibin (oractivin) present in this fluid.

Preparation of Antibody to α sub-unit (R1)

Human and Animal Fluid Samples

Pooled BFF was obtained from ovaries supplied by Midland Meat Packers,Crick, Northamptonshire. Ovaries were transported back to the laboratoryon ice and fluid aspirated within 4 hours of slaughter. Pooled HFF fromwomen undergoing hyperovolution treatment was obtained from Bourne HallClinic, Hourne, Cambridgeshire. Both fluids were stored in aliquots at-40° C. Human post-menopausal serum from a 63 year old woman wasobtained via the Blood Transfusion Laboratories, Oxford.

Synthetic Peptides and 32K Bovin Inhibin

As described in Groome et al HYBRIDOMA 9 31-42 (1990) synthetic peptidesused were based on published sequences near the amino terminal of the32K inhibin α subunit human 1-32 (for immunizations) was synthesizedusing pentafluorophenyl esters as described in a series of articles byAtherton and Sheppard and coworkers eg Eberley et al. (J. Chem. Soc.Perkin Trans. (1986) 361-367). Reagents for peptide synthesis wereobtained from Milligen U.K. Millipore House, 11-15 Peterborough Road,Harrow. The Pennisula peptides were not purified further. All thepeptides made here were purified by HPLC and the amino acid compositionchecked before use.

Purified bovin 12K inhibin was purchased from Pennisula Laboratories.

Coupling of Peptides to Carrier Proteins

Human 1-32 was coupled to tuberculin (PPD) via the cysteine using aheterofunctional agent. These couplings were carried out initially withreagents and protocols obtained from Cambridge Research BiochemicalsLtd, Button End, Harston, Cambridge. Tuberculin can be obtained from theCentral Veterinary Laboratory, Weybridge.

Immunization of Mice

Adult intact male Balb/c mice (Harlan-Olac Ltd. Station Road,Blackthorn, Bicester) were primed by one dose of human BCG vaccine(Glaxo) subcutaneously. One month later 50 μg of human peptide 1-32 as atuberculin conjugate was injected subcutaneously in Freund's incompleteadjuvant. Boosting with the same amount of immunogen in Freund'sincomplete adjuvant was given on four occasions at monthly intervals.The highest responding mice were assessed by ELISA and set aside forfusions. The fusion described here used a mouse that had received itslast boost 3 months previously.

Preparation of Monoclonal Antibodies

On each of the four days before fusion, 100 μg of immunogen was given insaline by intravenous injection into the tail vein. The spleen wasremoved and used for fusion of lymphocytes to SP2/0 myelomas by standardprotocols. The fusion was screened by ELISA on plates coated withporcine 1-32 peptide as described in a later section. Isotyping was doneusing a commercial isotyping kit based on red cell agglutination(Serotec Ltd). The antibody was purified from ascites by protein Achromatography. The clone R1 produced a mouse IgG2a and could also begrown in a hollow fibre bioreactor producing 2 mg/ml of antibodies.

Growth of hybridoma clones used for Inhibin Assay

Clone R1

Clone R1 producing antibody to the Inhibin α sub-unit. Spleen cellpatent was a Balb/c mouse. Myeloma line was SP2/0. Cells are routinelygrown in Iscove's Modified Dulbecco's medium (IMDM) obtained from Gibcocontaining 20% v/v foetal calf serum (Gibco) and 50 micrograms/mlgentamycin. Gas phase is 5% CO2 in air.

This clone grows well as mouse ascites producing about 2 mg ofantibody/ml of ascites or in a hollow fibre bioreactor producing up to 2mg/ml. The antibody is IgG2a.

Clone E4

Clone E4 is the beta-A sub-unit. Spleen cell parent was Balb/c mouse.Myeloma line was SP2/0. Cells are routinely grown in Iscove's modifiedDulbecco's medium containing 20% foetal calf serum (Gibco) and 50micrograms/ml gentamycin. Gas phase is 5% CO2 in air.

This clone is a low producer in mouse ascites (0.5 mg-1 mg/ml) but growswell in hollow fibre bioreactors producing up to 2 mg/ml. The antibodyis IgG2b.

Reagents and Procedures in accordance with the invention TWO SITE ENZYMEIMMUNOASSAY OF DIMERIC HUMAN INHIBIN IN FEMALE SERUM OR PLASMA

ASSAY PERFORMANCE

The antibodies needed for these assays are provided in a form whichenables the user to coat and use 20 microplates for the assay of dimericinhibin in human serum and plasma. This will enable the researcher toassay at least 800 serum/plasma samples. Further complete commercialassay kits are now available to the investigator (Serotec Ltd),supplying all the necessary preprepared reagents with detailedinstructions.

The assay can detect as little as 2 pg/ml of recombinant inhibin spikedinto human serum. Most post-menopausal women have less than 5 pg/ml,whereas following ovarian hyperstimulation levels can reach 1200 pg/ml.Throughout the normal cycle levels are in the approximate range 3-120pg/ml. Under optimal conditions the inter and intraplate coefficients ofvariation for sample duplicates are both less than 7%. Serial dilutionof samples containing high levels of immunoreactivity behave identicallyto recombinant 32 k inhibin diluted in postmenopausal serum.Quantitative recovery of spiked inhibin is observed both in thefollicular and luteal phase of normal cycles.

PROTOCOL FOR THE ENZYME IMMUNOASSAY OF DIMERIC INHIBIN IN HUMAN SERUMAND PLASMA

MATERIALS PROVIDED

1. Purified IgG from monoclonal antibody (E4) reacting with the beta-Asubunit of inhibin. 1 ml of a 1 mg/ml solution.

2. Fab-alkaline phosphatase conjugate of a monoclonal antibody (R1)reacting with the α subunit of inhibin. The optimum concentration ofthis conjugate varied between batches and was determined by trial anderror.

3. Recommended procedures, and step-by-step protocol.

NOT PROVIDED

1. Avidplate-HZ flat bottom. Obtained from Bioprobe International,14272, Franklin Avenue, Tustin, Calif. 92680, USA.

In the UK these plates can be obtained from Quantum Biosystems, 12Pembroke Avenue, Cambridge, CB5 9PB.

2. AMPAK kit for amplification of the alkaline phosphatase assay. 1 kitprovides assay reagents for 4 microplates. Obtained from DakoCorporation, 6392 Via Real, Carpinteria, Calif. 93013. In the UK thesekits can be obtained from Dako UK Ltd, 16 Manor Court Yard, HughendenAvenue, High Wycombe, Bucks UK.

3. Recombinant inhibin standard. The WHO is presently preparing astandard of recombinant human 32K inhibin and many inhibin workers willhave obtained such material for their previous work. A pool of serumfrom women undergoing ovulation induction could be used to preparestandards for in-house use, and this would simply need to be calibratedagainst a recombinant standard.

4. Hydrogen peroxide 30% (w/v). Use fresh analytical grade reagent.

5. A pool made of serum or plasma from post-menopausal women pretestedin the assay and shown to contain undetectable dimeric inhibin.Approximately half of all post-menopausal sera would be suitable.Samples pooled should be free of hemolysis. Fetal calf serum has alsobeen shown to be free from dimeric inhibin and may be used insubstituted for post-menopausal serum as a diluent.

COUPLING OF E4 ANTIBODY TO AVIDPLATES

This procedure is designed to minimise losses of the E4 antibody duringthe coupling procedure.

1. You are provided with 1 ml of E4 antibody at a concentration of 1mg/ml.

2. Transfer the solution to the minimum length of benzoylated dialysistubing (Sigma catalog no D2272 holds 10 ml/foot). Do not boil thedialysis tubing before use. Just wash it with water. We find losses ofantibody are lower with this tubing than conventional dialysis tubing.

3. Dialyze the antibody against three changes of 50 mM acetate buffer pH5.0 in the cold room.

4. Make up a 100 mM solution of sodium periodate (21 mg/ml).

5. Find out what volume of antibody solution you have after dialysis byweighing the liquid removed from the sac.

6. Add 0.1 volume of the periodate reagent to give a final concentrationof 10 mM.

7. Incubate the solution in the dark for 30 min at room temperature toallow aldehyde groups to be generated on the carbohydrate residues onthe Fc of the antibody.

8. Stop the reaction by adding 0.01 volume of ethylene glycol.

9. Desalt the solution back to 50 mM acetate buffer pH 5.0 on a short(15 ml bed volume) Sephadex G25 column. Detect the antibody as the firstpeak by reading fractions at 280 nm. Alternatively dialyze overnightagainst two changes of acetate buffer (1 liter) in the cold room for 24hours.

10. Read the final absorbance in a 1 cm cell. Antibody concentration inmg/ml will be absorbance/1.4.

11. Decide how much antibody in total you have and state this inmicrograms. At the end of this procedure you will probably have around600 micrograms of antibody. To coat the hydrazide plates we use aconcentration of 5 micrograms/ml. Thus you have enough to make 600/5=120ml of coating solution.

12. Add all the antibody to 120 ml of 50 mM acetate buffer pH 5.0 andmix thoroughly.

13. Avidplates-HZ wells are coated with 50 microliters overnight at roomtemperature. Place the plates in a box containing moist paper towellingto prevent evaporation during this procedure. You will have at leastsufficient reagent to coat 20-24 microplates. Complete recovery ofinhibin from serum matrix require a high density of functional E4antibody and we do not recommend using a lower concentration than thatstated here.

14. Make up a solution containing 0.1 M Tris-HCl pH 7.5 buffer, 1% BSA(Sigma protease free A3294), and 0.1% sodium azide.

15. One plate at a time, bang the plate dry on paper towelling and add150 microliters of the above solution.

16. Plates can now be stored in the cold room for periods of at least3-6 months and still give good results. Evaporation must be prevented.

17. Immediately before use the plate is washed on a plate washer with awash solution consisting of 0.05% (w/v) Tween 20, 0.15 M NaCl, in 0.05 MTris pH 7.5. (wash solution)

THE ASSAY PROCEDURE FOR HUMAN SERUM

MATRIX FOR STANDARDS

1. To dilute your standards and unknowns you will need some serum from apost-menopausal woman (PMS) or fetal calf serum, both of which are freefrom dimeric inhibin-A.

Note approximately half of all PMS sera have no detectable inhibin inthe assay described. Others give a low but significant signal (<5pg/ml). We are presently working to determine if this small signal isnon-specific or due to real inhibin. This is done by seeing if thesignal can be blocked by preincubating the Fab R1, alkaline phosphataseconjugate with the synthetic peptide immunogen used to make R1 beforeadding it to the well. If the signal can be blocked it is probably dueto inhibin. If not it may be due to heterophile antibodies in thatsample. Further information on this subject will be provided as itbecomes available.

FIND YOURSELF AN APPRECIABLE VOLUME OF SERUM GIVING AS LOW A SIGNAL, ASPOSSIBLE. This must be free of hemolysis.

INHIBIN FOR STANDARDS

Recombinant inhibin standards are made in the selected post-menopausalserum pool and stored at -80° C.

ASSAY DILUENT

5% (w/v) Triton X100 (Sigma X100)

10% (w/v) Bovine serum albumin (protease free, Sigma A3294)

5% (v/v) Normal mouse serum (Serotec Product code C1158)

All in 0.1 M Tris-HCL pH 7.5, 0.15 M sodium chloride.

Let it all stir on a stirrer for a few hours until it is clear.

Filter this solution through a 0.2 micron filter. It helps the filter ifyou remove the lumps by centrifugation or a prefilter (millex frommillipore).

Then add sodium azide to 0.1% (its safer than filtering azide) and storethe solution in the cold room. For longer term storage keep theunfiltered solution in the freezer, and filter and add azide in 100 mllots as needed.

ASSAY PROCEDURE

1. Make up standard recombinant inhibin concentrations in selectedpost-menopausal serum (PMS) from 500 pg/ml down to around 2 pg/ml.

2. Put 0.1 ml amounts of each standard or unknown sample in a small tubeand two 0.1 ml aliquots of just PMS in another two tubes.

It is important that serum or plasma samples from pregnant women arefree of hemolysis are obtained by gentle collection procedures.Excessive hemolysis will impair the action of the hydrogen peroxide, andgive underestimates of the amount of inhibin dimer. Similarly, thepost-menopausal serum used as a matrix for standards and a diluent forunknowns must also be free of hemolysis. Serum/plasma samples takenduring pregnancy will require dilution by approximately 10 told to bringthem into the accurate region of the standard curve. They are diluted inthe post-menopausal serum before the hydrogen peroxide treatment andthen processes as described for the standards.

3. Make up freshly a 5% (w/v) aqueous solution of hydrogen peroxide andadd 25 microliters to each tube. Mix gently. Oxygen bubbles will appearin the tube, sometimes more than others. This treatment is designed tooxidase the methionine residues in the epitope for the E4 antibody. Thisfollows an observation that the affinity of interaction of inhibin withthis antibody is increased by this method. It improves assay sensitivityby about 0 fold--8 fold depending on the state of oxidation of inhibinin the sample to start with. Note that samples and standards in theassay are both treated identically.

4. After a 30 min reaction period add 0.2 mls of assay diluent to eachtube. Mix.

5. Take an E4 coated hydrazide plate, (prepared as above) and wash itten times with 0.05% (w/v) TWEEN 20, 0.15 M NaCl, in 0.05 M Tris-HCl pH7.5. Bang the plate dry on tissue.

6. Without delay add 0.08 ml of each sample or standard to duplicatewells on the microplate.

Each plate must have a standard curve and precautions taken to make surethat evaporation or temperature gradients do not occur during the assay.Look out for any signs that the edge wells give abnormal readings.

7. Incubate the plate overnight in the cold room. We have it shaking butthis may not be essential.

8. Wash the plate 4 times with wash solution. Immediately add 50microliters of 1 in 400 dilution of the Fab R1 alkaline phosphataseconjugate.

9. Shake at room temperature for 1-2 hr. Make sure the plate is covered.

AMPLIFIED ELISA

Detection uses an amplification of the alkaline phosphatase reaction.However, equivalent results can be obtained without amplification usinga simple alkaline phosphatase substrate such as p-nitrophenyl phosphate.

PROCEDURE WITH AMPAK KIT

N.B. Thorough washing procedures are critical to the method.

1. After the incubation with the alk phos conjugated antibody above,wash the plate ten times on the plate washer. Hang dry.

2. Make up a 1 in 12 dilution in water of the AMPAK wash solution whichcomes with the kit. This is specially formulated to reduce non-specificbinding. We wash the plate twice more manually with this solutionallowing it to shake for two 15 minute periods in each.

Make sure the stock of this does not get in touch with alk phosconjugate.

3. Bang the plate and add kit substrate. (50 μl/well)

We make up all the kit substrate at one time. In order to allow thesubstrate reaction to run for longer we add additional magnesium to thesubstrate (1 μl/ml of 1 M MgCl1). Following advice from themanufacturers (Dako). We aliquot the substrate solution in 6 ml amountseach sufficient for 1 microplate, and store these frozen.

4. Cover the plate with parafilm and shake it at 37° C. for 2 h. Tosecure the parafilm we put a lid on the plate. It is important that atthis stage the plate is not subjected to temperature gradients, or theouter wells to evaporation.

5. Remove the plate from the incubator and allow it to return to roomtemperature for ten minutes.

6. Add 50 microliters of kit amplifier. This is also made up in advanceand stored frozen in 6 ml amounts. Because colour development is so fastthis must be added to the wells in a timed sequence (eg 1 row each 5sec). When all the wells have had amplifier added tap the plate gentlyto mix and allow it to stand for 3-10 minutes until the post-menopausalwells start to develop visible colour.

7. Stop the reaction by adding 50 microliters of kit stop solution inthe same timed sequence as amplifier.

8. Read the plate at 490 nm with a reference wavelength of 620 nm. MostELISA readers will plot your standard curve.

9. Serum samples from hyperovulated or pregnant women can have more than1000 pg/ml and will need to be diluted with PMS into the accurate regionof the standard curve.

10. The plates are easy to photograph and can be stored frozen if theycannot be read or photographed directly.

ASSESSMENT OF ASSAY PERFORMANCE

1. Excellent recovery of recombinant 32 k inhibin spiked into humanserum and plasma or even blood.

2. Works for both serum and heparinised plasma with insignificantdifference in levels.

3. Samples from hyperovulated women dilute out closely parallel to the32 k standards in PMS as do samples from women at the peak of the lutealphase in a normal cycle.

4. The assay has been validated for human female samples.

5. Activin and tree α subunits have less than 0.1% cross-reactivity inthe assay following the procedures described.

6. A typical specimen set of results for the assay of recombinantstandards diluted in postmenopausal serum is shown below in Table 1.

    ______________________________________    Concentration of recombinent standard    in serum from post-menopausal woman                             Estimated concentration    Picograms/ml              Absorbances    from fitted standard curve    ______________________________________    0         0.057, 0.53, 0.051, 0.057    2         0.091, 0.089    3.9       0.117, 0.122   4.3    7.8       0.170, 0.169   8.3    15.6      0.281, 0.294   16.4    31.21     0.496, 0.510   30.2    62.5      0.925, 0.935   62.5    125       1.387, 1.434   125    ______________________________________

The invention will no be further described by comparing the priormethods of assay in both the first and second trimester with that of thepresent invention.

First Trimester

Previously, we have studied two different immunoreactive inhibin assaysrespect to the detection of Down's syndrome in the first trimester. Oneused an antibody raised against 31 kD bovine inhibin and the other, acommercial two-site assay, using two antibodies directed against 2distinct α-sub-unit epitopes. The first assay was a heterologousradioimmunoassay using an antibody (1989) raised against 31 kDa bovininhibin and a tracer of iodinated 31 kDa bovine inhibin. Recombinanthuman inhibin-A (rhINH-R-90/1) was used for standards with the resultsexpressed as pg/ml. The sensitivity of the assay was 780 pg/ml and thecoefficients of variation of intra-assay and inter-assay were 6.6% and11.5% respectively. This assay is now distributed by the NationalInstitute of Child Health and Human Development (NICHD).

The second assay, a commercial solid-phase two site immunoenzymaticassay (Medgenix, High Wycombe UK) was used to assay the same samples.The two antibodies employed in this assay were directed against distinctepitopes of the a-subunit of human inhibin. Human inhibin was used asstandards and the results were expressed as U/ml. The sensitivity of theassay was 0.1 U/ml and the coefficients of variation of intra-assay andinter-assay were 1.9% and 8.9% respectively.

These were evaluated using maternal serum from 11 women with anidentified Down's affected pregnancy and 44 controls (4 matched to eachDown's sample by gestation and duration-of-storage). 5 of the Down'ssamples had been collected at 11 weeks and 6 at 12 weeks.

Using the NICHD Assay, the mean (±SEM) maternal serum immunoreactiveinhibin at 11 and 12 weeks gestation in the Down's syndrome samples were3186±195 pg/ml and 2517±441 pg/ml and in the controls 2020±172 pg/ml and2561±198 pg/ml respectively. The mean immunoreactive inhibin levels atthe two gestations were not significantly different for either group,and there were no significant differences between Down's syndrome andnormal pregnancies (2821±267 pg/ml and 2317±138 pg/ml respectively,n.s.).

Using the Medgenix Assay, the mean (±SEM) maternal serum immunoreactiveinhibin at 11 and 12 weeks gestation in the Down's syndrome samples were2.46±0.56 U/ml and 2.35±0.62 U/ml and in the controls 1.84±0.17 U/ml and2.20±0.33 U/ml respectively. The mean immunoreactive inhibin levels atthe two gestations were not significantly different for either group,and there were no significant differences between Down's syndrome andnormal pregnancies (2.4±0.4 U/ml and 2.04±0.19 U/ml respectively, n.s.).

Further, there was poor correlation between the inhibin levels derivedfrom the two assays.

In conclusion statistical analysis of the data revealed that neitherassay detected a significant effect of gestation on serum inhibinlevels. After combining the data from both gestations, no significantdifference between the Down's samples and controls for either assay wasdetected. These data suggest that these two assays detect differentinhibin species in pregnancy serum but that neither will be useful forthe detection of Down's syndrome in the late first trimester. Incontrast, we have now established that inhibin-A is an extremely goodmarker for Down's syndrome in the first trimester.

23 women were identified from records of known Down's affectedpregnancies allowing their stored serum to be retrieved. The sera from 8of these women had been collected at 11 completed weeks of gestation, 8at 12 completed weeks and 7 at 13 completed weeks. These gestations hadbeen calculated from ultrasound scans performed on the day of sampling.Similarly, for each Down's affected sample 4 control women, matched forgestation (ultrasound determined) and duration of storage, wereidentified and their samples retrieved. Three control samples, one eachmatched for three different 11 week Down's samples, were unable to beused due to insufficient sample volume, making 89 control samplesavailable for assay.

In these samples from the first trimester the mean (95% CI) maternalserum dimeric inhibin A in the control samples was 389.5 (341.3-437.7)pg/ml, 341.3 (279.9-402.7) pg/ml and 263.6 (218.7-308.4) pg/ml at 11, 12and 13 weeks respectively. In the Down's syndrome samples the median MoMwas 2.46 (95% CI 2.11-3.26) and for a given FPR of 5%, 65% (15/23) ofthe Down's Syndrome samples were detected (Table 1). FIG. 1 shows the10th, 50th and 90th centiles for the 23 Down's syndrome cases plotted asMoMs.

The novel data presented indicate that maternal serum dimeric inhibin-Ais a useful serum marker of Down's syndrome in the first trimester ofpregnancy.

Second Trimester

In the second trimester, only the Medgenix assay has been evaluated.These data have been alluded to earlier. In the first report (van Lithet al 1992) it was shown that immunoreactive inhibin was elevated inassociation with Down's syndrome. Subsequently, one other report hasconfirmed this. However, it was suggested that the wide distribution ofinhibin levels in both the control and Down's syndrome pregnancies madeit unlikely that inhibin, as detected, would be a valuable marker.

This invention details data showing for the first time that, as in thefirst trimester, in the second trimester inhibin-A is significantlyelevated to a degree that would offer high detection rates of Down'ssyndrome. In 21 Down's syndrome pregnancies at 15-17 weeks, theinhibin-A median MoM was 2.6, calculated from 150 normal pregnancies(FIG. 2). It was calculated that for a given FPR of 5.3% a detectionrate of 62% would be afforded (Table 2).

Thus, in both the first and second trimesters, elevated levels ofdimeric inhibin-A have been shown to be an indication of Down'ssyndrome. The levels required to indicate positive results may befurther refined as development of the test proceeds. The increase is amarked increase e.g. up to twice the levels of normal pregnancies. Theseresults are very different from those reported on the other non-specificinhibin assays, both in the first and second trimesters.

                  TABLE 1    ______________________________________    Number (percentage) of affected and unaffected    pregnancies in the first trimester (11-13 weeks) with a    given MoM above different arbitrary levels.            Number (%) of Number (%) of    MoM     Affected Pregnancies                          Unaffected Pregnancies    ______________________________________    0.5      23 (100)     86 (97)    1.0     22 (96)       42 (47)    1.5     19 (83)       18 (20)    2.0     15 (65)       6 (7)    2.5     10 (43)       1 (1)    3.0      9 (39)       1 (1)    3.5      5 (22)       0 (0)    4.0      4 (17)       0 (0)    4.5      3 (13)       0 (0)    ______________________________________

                  TABLE 2    ______________________________________    Number (percentage) of affected and unaffected    pregnancies in the second trimester (15-17 weeks) with a    given MoM above different arbitrary levels.            Number (%) of Number (%) of    MoM     Affected Pregnancies                          Unaffected Pregnancies    ______________________________________    0.5     21 (100)      135 (90)    1.0     21 (100)      76 (51)    1.5     21 (100)      28 (19)    2.0     14 (67)       11 (7)    2.5     11 (52)       4 (3)    3.0     7 (33)        1 (1)    3.5     6 (29)        0 (0)    4.0     6 (29)        0 (0)    4.5     4 (19)        0 (0)    ______________________________________

Legends to Figures

FIG. 1. The 10th, 50th and 90th percentiles of maternal serum inhibin-AMoMs of 89 chromosomally normal pregnancies with levels from 23individual Down's affected pregnancies.

FIG. 2. The 10th, 50th and 90th percentiles of maternal serum inhibin-AMoMs of 150 chromosomally normal pregnancies with levels from 21individual Down's syndrome pregnancies.

We claim:
 1. A method of testing for indications of Down's syndromewhich comprises measuring the level of inhibin-A in a sample of maternalbody fluid taken during gestation, which inhibin-A comprises anα-sub-unit and a β-sub-unit (βA).
 2. A method according to claim 1,which is specific for Inhibin-A.
 3. A method according to claim 1, inwhich said method comprises the use of an antibody (βA antibody)specific for the inhibin-A β sub-unit (βA sub-unit).
 4. A methodaccording to claim 3 in which the βA antibody is a monoclonal antibody,said monoclonal antibody is derived from a hybridoma, which is preparedusing a peptide corresponding to a part of the βA sub-unit as animmunogen.
 5. A method according to claim 4, in which the peptide is asynthetic peptide corresponding to part of a C-terminal region of the βAsub-unit.
 6. A method according to claim 3, in which the βA antibody isused to capture inhibin-A from a test sample.
 7. A method according toclaim 1, in which the method comprises the use of an antibody specificfor Inhibin-A α sub-unit.
 8. A method according to claim 7, in which αsub-unit antibody is derived from a hybridoma, which is prepared using apeptide corresponding to a part of the inhibin-A α sub-unit as animmunogen.
 9. A method according to claim 8, in which the peptide is asynthetic peptide corresponding to part of an N-terminal region of the αsub-unit.
 10. A method according to claim 8, in which a Fab fragment ofthe antibody is used.
 11. A method according to claim 7, in which the αsub-unit antibody is used as a detection antibody and is linked to adetectable marker.
 12. A method according to claim 11, in which thedetection antibody is enzyme-linked.
 13. A method according to claim 1,in which the maternal body fluid is serum or plasma.
 14. A methodaccording to claim 1, carried out in the first trimester of gestation.15. A method according to claim 14, carried out from the eleventh to thethirteenth week of gestation.
 16. A method according to claim 1, carriedout in the second trimester of gestation.
 17. A method according toclaim 1, carried out as a screening test to select patients forsubsequent diagnostic testing.
 18. A method according to claim 17,carried out as an adjunct to the assay of other markers.
 19. A reagentkit for screening for Down's Syndrome comprising a first antibodyspecific for a β sub-unit of inhibin-A and a second antibody specificfor an α sub-unit of inhibin A.
 20. A reagent kit according to claim 19,in which the first antibody is attached to a support material.
 21. Areagent kit according to claim 19, in which the second antibody islinked to a detectable marker.